Pulsed EPR and DFT Characterization of Radicals Produced by Photo

1806

J. Phys. Chem. B 2008, 112, 1806-1819

Pulsed EPR and DFT Characterization of Radicals Produced by Photo-Oxidation of Zeaxanthin and Violaxanthin on Silica-Alumina A. Ligia Focsan,† Michael K. Bowman,† Tatyana A. Konovalova,† Pe´ ter Molna´ r,‡ Jozsef Deli,‡ David A. Dixon,*,† and Lowell D. Kispert*,† Department of Chemistry, UniVersity of Alabama, Tuscaloosa, Alabama 35487-0336, and Department of Medical Chemistry, UniVersity of Pe` cs, H-7601, Pe` cs, Hungary ReceiVed: August 15, 2007; In Final Form: October 24, 2007

Pulsed electron nuclear double resonance (ENDOR) and two-dimensional (2D)-hyperfine sublevel correlation spectroscopy (HYSCORE) studies in combination with density functional theory (DFT) calculations revealed that photo-oxidation of natural zeaxanthin (ex Lycium halimifolium) and violaxanthin (ex Viola tricolor) on silica-alumina produces the carotenoid radical cations (Car•+) and also the neutral carotenoid radicals (#Car•) as a result of proton loss (indicated by #) from the C4(4′) methylene position or one of the methyl groups at position C5(5′), C9(9′), or C13(13′), except for violaxanthin where the epoxide at positions C5(5′)-C6(6′) raises the energy barrier for proton loss, and the neutral radicals #Car•(4) and #Car•(5) are not observed. DFT calculations predict the largest isotropic β-methyl proton hyperfine couplings to be 8 to 10 MHz for Car•+, in agreement with previously reported hyperfine couplings for carotenoid π-conjugated radicals with unpaired spin density delocalized over the whole molecule. Anisotropic R-proton hyperfine coupling tensors determined from the HYSCORE analysis were assigned on the basis of DFT calculations with the B3LYP exchangecorrelation functional and found to arise not only from the carotenoid radical cation but also from carotenoid neutral radicals, in agreement with the analysis of the pulsed ENDOR data. The formation of the neutral radical of zeaxanthin should provide another effective nonphotochemical quencher of the excited state of chlorophyll for photoprotection in the presence of excess light.

Introduction Carotenoids act as accessory light-harvesting pigments in photosynthetic antenna pigment-protein complexes, efficiently transferring energy to nearby bacteriochlorophylls.1-4 They also assume a number of photoprotective roles related to their ability to quench chlorophyll triplet states, thereby preventing the formation of harmful singlet oxygen.4-6 In addition, the carotenoid cofactor in the photosystem II (PSII) reaction center can act as an alternative electron donor to reduce P680+ under conditions where the primary electron-transfer pathway is blocked and, in such a way, prevent damage to reaction centers of oxygenic photosynthetic organisms.7-11 The carotenoid radical cation (Car•+) can be stoichiometrically generated by illumination of Mn-depleted PSII at 20 K.12 Warming to 120-200 K resulted in reduction of Car•+ via electron transfer from ChlZ forming ChlZ•+.12,13 The Car•+ and ChlZ•+ radicals of PSII have been extensively studied by optical,9,14 Raman,15-17 FTIR,18 X-band (9 GHz),12,13 and highfrequency (130, 285 GHz) electron paramagnetic resonance (EPR) spectroscopy.19,20 EPR signals of Car•+ and ChlZ•+ are not distinguishable in X-band spectra. They have a quite similar broad unresolved line shape ∼14 wide G and are centered at g ∼ 2.0027.12,13 To identify the nature of the carotenoid radical and gain more information on the electronic structure of Car•+, high-frequency EPR studies have been used to resolve the Car•+ * Corresponding authors. E-mail: [email protected] (D.A.D.) and [email protected] (L.D.K.). † University of Alabama. ‡ University of Pe ` cs.

g tensors in PSII (130 GHz, 285 GHz)19,20 and in the model system: canthaxanthin on silica-alumina support (330-670 GHz).21 Further characterization of the Car•+ hyperfine structure is deduced from electron nuclear double resonance (ENDOR) spectroscopy. Continuous wave (CW) and pulsed ENDOR spectroscopies have provided information on the nearly isotropic β-proton hyperfine couplings of Car•+ in PSII,22 in organic solutions23 and on solid supports.24-28 In addition, twodimensional hyperfine sublevel correlation spectroscopy (2DHYSCORE) has been used to determine highly anisotropic R-proton hyperfine tensors of the Car•+ in PSII29 and on a silica-alumina support.27 Isotropic β-methyl proton hyperfine coupling observed from CW ENDOR measurements for Car•+ in PSII was on the order of 8-9 MHz.22,29 This was consistent with density functional theory (DFT) calculations30 for all-trans β-carotene conformation, which showed that the unpaired spin density in Car•+ is delocalized over the carotenoid π-conjugated system resulting in isotropic β-proton methyl hyperfine couplings smaller than 9 MHz. We have found similar results in DFT studies of the radical cation of β-carotene.31 By using different DFT functionals and basis sets, it was confirmed that the isotropic β-methyl proton hyperfine couplings do not exceed 9 MHz for the methyl groups on the carotenoid radical cation. These methyl groups undergo rapid rotation even at 5 K, and thus powder CW ENDOR lines for these couplings are resolved and detectable. DFT calculations31 of neutral carotenoid radicals (#Car•) formed by loss of H+ (loss of H+ is indicated by #) from a methyl group of the radical cation provided an explanation for

10.1021/jp0765650 CCC: $40.75 © 2008 American Chemical Society Published on Web 01/19/2008

Photo-Oxidation of Zeaxanthin and Violaxanthin SCHEME 1: Carotenoid Structures

the 13 MHz isotropic couplings observed by ENDOR measurements for methyl protons in UV irradiated carotenoids supported on silica-alumina matrices or Nafion films.28 It was predicted31 that these methyl proton couplings were due to a neutral carotenoid radical formed by loss of H+ upon photolysis from the C5(5′)-methyl group of Car•+ (Aiso ) 12.5 MHz), leading to the most stable radical; loss of H+ from the methyl groups at positions C9(9′) (Aiso ) 13.5 MHz) and C13(13′) (Aiso ) 16 MHz) is also possible, but the resulting isomers are less stable by 5.4 kcal/mol and 1.9 kcal/mol, respectively, than the most stable structure. The original assignment of the 13 MHz proton couplings to the C13(13′)-methyl group was based on RHFINDO/SP calculations, the best available at the time.25 Lack of a powder ENDOR detectable 13 MHz coupling for the carotenoid radical cation formed on activated silica-alumina in the absence of UV photolysis25 was attributed to the influence of the matrix preventing rapid rotation of the C13-methyl proton, causing the powder ENDOR spectrum to be broadened and the 13 MHz powder ENDOR line not to be observable. It has now been shown25,31 that the neutral carotenoid radical is not formed in absence of UV photolysis, and thus, no resolvable methyl proton coupling of 13-16 MHz would have been observed. Here, we apply the methods of pulsed ENDOR and 2DHYSCORE spectroscopies to characterize carotenoid radicals of zeaxanthin (ex Lycium halimifolium) and violaxanthin (ex Viola tricolor; see Scheme 1) photogenerated on silica-alumina. Zeaxanthin (Zea) is a key component involved in energy dissipation in PSII. In excess light and at pH < 6, Zea is produced from violaxanthin (Vio) in thylakoid membrane via the xanthophyll cycle.32 In limiting light and pH > 6, the equilibrium mixture favors Vio. Femtosecond transient absorption measurements demonstrated that deactivation of 1Chl* during excess light occurs by excitation transfer to a Chl-Zea heterodimer, followed by ultrafast Zea•+ formation.33 Experimental Section Zea and Vio were extracted and purified in Molna´r’s laboratory as described earlier.34-36 Samples of 0.8 mL of 5 × 10-3 M carotenoid solutions in CH2Cl2 were degassed in quartz EPR tubes (WILMAD, o.d. 4 mm) by three freeze-pumpthaw cycles. A sample of 0.08 g of silica-alumina (Aldrich) activated at 550 °C for 3 h and cooled in air at room temperature (RT) was added to the carotenoid solution contained in an EPR tube. The solvent was evaporated under reduced pressure, and the tube was evacuated and sealed. The samples were irradiated at λ ∼ 350 nm at 77 K with a 200 W Xe lamp and stored at 77 K before use. Irradiation was done at a distance of 10 cm for 2 to 10 min duration. X-band pulsed ENDOR and 2D-HYSCORE experiments were carried out with a Bruker E-580 EPR spectrometer using

J. Phys. Chem. B, Vol. 112, No. 6, 2008 1807 SCHEME 2: Loss of H+ upon the Photolysis of the Zea•+

a Bruker EPR MD5-W1 cavity. The spectrometer is controlled through the LINUX workstation with Xepr, the BRUKER EPR acquisition and data manipulation software. The temperature of the cavity is maintained with a liquid-He cryostat (CF935) with electrically controlled OXFORD He transfer line. Microwave pulses were applied with a 20 W TWT amplifier. Pulsed ENDOR spectra were recorded with a Bruker DICE pulsed ENDOR accessory, an ENI A-500 RF power amplifier using the Davies (π-T-π/2-τ-π-τ-echo) and Mims (π/2-τ-π/2-T-π/ 2-τ-echo) pulse sequences where the additional RF π pulse is applied during the separation time T. Hyperfine sublevel correlation spectroscopy (HYSCORE), a two-dimensional (2D) four-pulse electron spin echo envelope modulation (ESEEM) technique, which provides correlations between nuclear frequencies originating from different electron manifolds, was employed. 2D-HYSCORE (π/2-τ-π/2-t1-π-t2π/2-τ-echo) spectra were recorded where the echo amplitude was measured as a function of t1 and t2.37 Pulsed ENDOR hyperfine coupling simulations were performed using the SimBud/SpecLab programs.38 DFT Calculations. All calculations were done with the Gaussian 03 program package39 on the Cray XD1 computer at the Alabama Supercomputer Center. Geometries were optimized at the DFT level with the B3LYP exchange-correlation functional40 and the 6-31G** basis set,41 which we have previously shown is reasonable for predicting the geometry of β-carotenebased radicals.31 Single point calculations on these geometries were used to predict ENDOR and 2D-HYSCORE hyperfine couplings at the B3LYP level with the TZP basis set from the Ahlrichs group.42 This basis set has been shown to give good

1808 J. Phys. Chem. B, Vol. 112, No. 6, 2008

Focsan et al.

Figure 1. Unpaired spin distribution for zeaxanthin radicals (A) Zea•+, (B) #Zea•(4), (C) #Zea•(5), (D) #Zea•(9), and (E) #Zea•(13) from DFT calculations. The blue represents excess R and the green excess β unpaired spin density. The carbon atoms are gray, the oxygen atoms are red, and the hydrogen atoms are white.

NMR chemical shifts43 and good EPR parameters for carotenes.31 The unpaired spin densities were obtained using the AGUI interface44 from the wave-functions and spin densities produced by Gaussian 03. The spin density is defined as the difference in the alpha and beta spin densities.

Results and Discussion DFT Calculations. Zea and Vio are symmetric molecules which have six methyl groups in three distinct positions: C5(5′), C9(9′), and C13(13′) (see Scheme 1). The prime

Photo-Oxidation of Zeaxanthin and Violaxanthin

J. Phys. Chem. B, Vol. 112, No. 6, 2008 1809

Figure 2. Unpaired spin distribution for violaxanthin radicals (A) Vio•+, (B) #Vio•(4), (C) #Vio•(5), (D) #Vio•(9), and (E) #Vio•(13) from DFT calculations. See Figure 1 caption.

positions of these molecules are equivalent by symmetry with the unprime positions, so calculations of the neutral carotenoid radicals (#Car•) were carried out only for the unprimed positions. Calculations were carried out for the radical cations Zea•+ and Vio•+ and for the #Zea•(4), #Zea•(5),

#Zea•(9), #Zea•(13), #Vio•(4), #Vio•(5), #Vio•(9), and #Vio•(13) neutral radicals, which were expected to occur upon light irradiation of Zea (see Scheme 2) and similarly of Vio on activated silica-alumina on the basis of the results for β-carotene.31


Focsan et al.

TABLE 1: Isotropic β-Methyl Proton Hyperfine Couplings Aiso (MHz) of Zea Radicals from DFT Calculations Used to Simulate the Pulsed ENDOR Powder Spectra in Figure 3a DFT calculated Aiso position

Zea•+

#Zea•(4)

#Zea•(5)

#Zea•(9)

#Zea•(13)

C1-CH3

-0.09 0.07 -0.02 -0.04 5.69 C 6.62 D 8.33 D 8.26 D 5.12 C 4.86 C

∆E ) 0 0.67 -0.01 -0.01 0.03 -3.00 B 2.01 B -7.23 D 7.81 D -8.71 D 11.84 F

∆E ) 3.2 0.10 0.58 0.04 -0.01

∆E ) 8.4 0.02 0.04 0.04 -0.01 -0.40 A 2.65 B

∆E ) 10.2 -0.002 -0.02 0.05 -0.02 -0.22 A 3.50 B -1.19 A 12.20 F

C1′-CH3 C5-CH3 C5′-CH3 C9-CH3 C9′-CH3 C13-CH3 C13′-CH3

2.60 B -4.47 C 9.91 E -7.35 D 14.06 G

9.97 E -8.23 D 14.00 G

16.10 H

a

Letters A to H indicate the peaks in Figure 3 corresponding to the isotropic couplings. The hyperfine coupling tensors for #Zea•(4) are nearly identical to those calculated for #Zea•(5), #Zea•(9), and #Zea•(13); however, note the absence of the isotropic 13-16 MHz couplings. ∆E ) relative energies for the neutral radicals in kilocalories per mole.

TABLE 2: Isotropic β-Methyl Proton Hyperfine Couplings Aiso (MHz) of Vio Radicals from DFT Calculationsa DFT calculated Aiso position

Vio•+

#Vio•(4)

#Vio•(5)

C1-CH3

0.06 0.10 0.04 0.23 0.26 A 0.23 A 9.93 C 10.0 C 6.07 B 6.03 B

∆E ) 15.3 -0.03 0.29 0.0001 0.004 -1.4 0.002 -0.87 0.23 -0.49 0.42

∆E ) 19.4 0.09 0.53 0.00003 0.001

C1′-CH3 C5-CH3 C5′-CH3 C9-CH3 C9′-CH3 C13-CH3 C13′-CH3

0.0008 -0.32 0.09 -0.18 0.15

#Vio•(9) #Vio•(13) ∆E ) 0 -0.004 0.01 0.004 0.14 -0.02 A 0.07 A

∆E ) 1.8 -0.003 -0.01 0.004 0.18 -0.01 A 0.09 A -1.38 A 12.08 D

9.30 C -8.55 C 13.74 D 16.38 E

a Hyperfine couplings of Vio•+, #Vio•(9), and #Vio•(13) were used to simulate the pulsed ENDOR powder spectra in Figure 4. The letters A to E indicate the peaks in Figure 4 corresponding to the isotropic couplings. ∆E ) relative energies for the neutral radicals in kilocalories per mole.

The optimized geometry parameters for the minimized structures are given in Supporting Information (Tables S1-S10). The unpaired spin density distributions are shown pictorially in Figures 1 and 2. Notably, when a proton is lost from the radical cation (as in Scheme 2), the unpaired spin density increases at carbons along the chain distant from the C4 methylene group or the methyl groups (Figures 1B-E, 2D, 2E) where the proton was lost upon light irradiation. The exceptions are #Vio•(5) and #Vio•(4) where the unpaired spin density is localized on the C5, C6 atoms and C4 atom, respectively. The DFT isotropic β-proton couplings are given in Table 1 and 2, while the anisotropic R-proton couplings are given in Tables 3, 4 and 5. The reason for this grouping is as follows: previous CW ENDOR measurements25,28 showed resolved proton ENDOR lines which were attributed to the presence of rapidly rotating methyl groups. Subsequently, it was shown21 that the β-methyl protons in carotenoids undergo rapid rotational motion even at 5 K which averages out the small dipolar anisotropy and results in the same isotropic proton coupling for all three protons in each methyl group in the ENDOR spectrum. This is the reason that β-methyl protons in carotenoid radicals give rise to narrow, well resolved and intense CW ENDOR lines in powder spectra.45 According to our previous data on carotenoid radicals, CW ENDOR lines from the methyl protons are wellresolved, while lines from R-protons are often broadened and

not easily detected.23-26 The magnitudes of the hyperfine coupling constants obtained for the carotenoid radicals in this work are quite similar to those measured previously for the methyl protons at positions C5(5′), C9(9′), and C13(13′) of Car+• on solid support including silica-alumina24-27 and also calculated by RHF-INDO/SP methods.24,26 The anisotropic R-proton couplings were never resolved and contributed only to the broad back-ground signals. Thus the calculated couplings were grouped into tables showing the isotropic and anisotropic couplings to emphasize the significant contribution of the methyl protons to the resolved lines of the ENDOR spectrum. Significantly, the isotropic β-methyl proton couplings larger than 9 MHz (E to H in Table 1 and C, D, and E in Table 2) are only predicted for the neutral radicals. In addition, loss of a proton from the C5(5′) position of the radical cation in Vio (Figure 2A) generates a vinyl radical where the unpaired spin density is localized at the C5(5′) carbon atom (Figure 2B). This structure is 19.4 kcal/mol higher in energy than the most stable radical formed by proton loss. This vinyl radical exhibits large (-92, -57, -21 MHz) anisotropic couplings (Table 5) which are not experimentally observed in the EPR spectrum, consistent with the high energy of the radical. Proton loss from the C4(4′) position forms a structure 15.3 kcal/mol higher in energy, with a radical localized at the C4(4′) position and isotropic couplings as high as -54.7 and 75.8 MHz (Table 5), which are not experimentally observed in the EPR spectrum. However, loss of a H+ from the methyl group at the C9(9′) or C13(13′) position for Vio exhibits similar hyperfine splittings as for Zea. Pulsed EPR Measurements. Echo-induced field swept EPR spectrum (Supporting Information, Figure S1) of the Zea radicals adsorbed on silica-alumina exhibits a signal centered at g ∼ 2.0028 and about 10-14 G wide which is characteristic of a π-radical Car•+. The two-pulse ESEEM spectrum of the zeaxanthin radicals measured at the field position corresponding to the maximum EPR absorption exhibits proton modulation only (Supporting Information, Figure S2). Pulsed ENDOR Measurements. The powder Davies 1H ENDOR spectrum recorded after reaction of Zea on silicaalumina at the maximum EPR absorption consists of two broad lines, each composed of many overlapping lines spaced symmetrically around the free proton frequency (Figure 3a). It is not possible to deconvolute the poorly resolved spectrum. However, DFT calculated hyperfine coupling tensors have been shown31 to be accurate enough to use for the simulation of the spectrum (Figure 3b) if both the isotropic β-methyl proton and anisotropic R-proton tensors (Table 1, 3 and 4) are used for all five Zea radical species. If the pulsed ENDOR spectrum is simulated using the calculated DFT isotropic couplings due to the β-methyl protons for the Zea•+, #Zea•(4), #Zea•(5), #Zea•(9), and #Zea•(13) (Table 1), then the resulting spectrum (Figure 3c) exhibits peak maxima indicated by the letters A, B, C, D, E, F, G and H. These peak maxima are broadened upon addition of the anisotropic tensors in Figure 3b. If the DFT isotropic and anisotropic couplings for just the radical cation (Tables 1 and 3) are used, then the ENDOR spectral peaks (Figure 3d) at positions F, G, and H are not accounted for. Similarly, for Vio (Figure 4a), the powder 1H ENDOR spectrum exhibits resolved features which can only be simulated (Figure 4b) if the full tensor (anisotropic and isotropic components) found in Tables 2, 3, and 5 for the three species (Vio•+, #Vio•(9), and #Vio•(13)) are utilized. The fit at point D and E is especially crucial as it requires contributions from #Vio•(9) (point D) and #Vio•(13) (point E) as well as contributions from line C due to #Vio•(9). Figure 4c shows the fit if only isotropic



TABLE 3: Anisotropic r-Proton Tensor Components AXX, AYY, and AZZ (MHz) of Radical Cations from DFT Calculationsa Zea•+ position

Vio•+

AXX

AYY

AZZ

Aiso

1.2 -0.17 -0.07 -0.32 -0.67 -0.67 -0.55 -0.24 13.1 2.82 13.09 9.2

1.3 -0.05 0.01 -0.23 -0.61 -0.28 -0.43 -0.21 13.49 2.87 13.34 9.48

1.77 0.63 0.52 0.64 0.86 0.96 0.53 0.18 14.72 4.34 15.03 10.96

1.42 0.14 0.15 0.03 -0.14 0.003 -0.15 -0.09 13.77 3.34 13.82 9.88

C5 C7

-15.29

-10.77

-4.75

-10.27

C7′

-15.53

-10.78

-4.65

-10.32

C8

1.71 [-1.58] 1.76 [-1.58]

C2 C2′ C3 C3′ C4 C4′

C8′

2.33 2.36

5.47 [6.34] 5.77 [6.34]

3.17 3.3

C9 C10 C10′ C11

0.73 0.62 -11.6

1.35 1.25 -8.57

3.88 3.77 -3.58

1.99 1.88 -7.92

C11′

-11.32

-8.38

-3.47

-7.72

1.17 1.01

4.0 3.76

1.99 1.79

C12 C12′ C13 C14 C14′ C15

0.8 0.6 -2.09 -2.38 -4.66

-1.94 -2.3 -4.02

0.79 0.57 -0.77

-1.08 -2.84 -3.15

C15′

-4.23

-3.71

-0.58

-2.84

AXX 1.08 -0.18 0.63 -0.229 -0.27 -0.1 -0.21 -0.005 -0.11 1.46 0.11 3.03 -15.41 [-14.1] -15.38 [-14.1] 1.92 1.88 1.29 1.23 -13.68 [-14.1] -13.67 [-14.1] 1.19 1.12 -2.29 -2.38 -5.21 [-5.06] -5.10 [-5.06]

AYY

AZZ

Aiso

1.15 -0.16 0.69 -0.199 -0.21 -0.087 -0.18 -0.01 -0.005 1.6 0.16 3.21

1.59 0.43 1.15 0.426 0.32 0.175 0.33 -0.04 0.62 2.14 0.94 3.7

1.27 0.03 0.82 -0.001 -0.05 -0.004 -0.02 -0.02 0.17 1.73 0.40 3.31

-10.61 -10.63 2.68 [3.16] 2.66 [3.16] 1.9 1.86 -10.07 -10.07 1.56 1.51 -2.09 -2.19 -4.52 [-3.16] -4.44 [-3.16]

-4.28 [-5.54] -4.23 [-5.54] 4.89 [5.06] 5.54 [5.06]

-10.1 -10.08 3.39 3.36

4.91 4.86 -4.27 [-5.54] -4.25 [-5.54] 4.84 4.78

2.70 2.65 -9.34 -9.33

0.93 -0.87 -0.87

-1.15 -1.23 -3.53

-0.81

-3.45

2.53 2.47

a HYSCORE couplings (in square brackets) were assigned to the tensors obtained by DFT. The bold values are the isotropic coupling constants Aiso given by averaging the three anisotropic coupling tensors AXX, AYY, and AZZ.

proton couplings from methyl groups of the three species (except #Vio•(4) and #Vio•(5)) are used. This fit is not as good as when the full isotropic and anisotropic tensors are incorporated. If the isotropic and anisotropic couplings (Tables 2 and 3) are used for only the radical cation (Figure 4d), then the D to E experimental region is not accounted for and no agreement with experiment is found. As discussed above, loss of a proton from the C4(4′) and C5(5′) positions is energetically unfavorable and not observed, so loss from these positions does not occur and was not used for simulation. The Mims ENDOR spectra are given in the Supporting Information (Figures S3 and S4). The pulse delay time of 108 ns eliminates observation of couplings of the order of 5 MHz so simulation using the full R- and β-hyperfine coupling tensors were not done. However, the Mims ENDOR spectrum does show large couplings between 12 and 16 MHz from the methyl groups of the neutral radicals (couplings F, G, and H for #Zea• in Figure S3 and couplings D and E in Figure S4 for #Vio•). These large couplings generate the Mims ENDOR lines above 21 MHz. 2D-HYSCORE Measurements. Previously, it had been shown27 that the anisotropic R-proton tensors could be deduced from HYSCORE measurements. Since DFT calculations provide the anisotropic R-proton couplings (Tables 3, 4, and 5), a comparison can be made between the DFT anisotropic tensors and those determined experimentally from HYSCORE measurements. From such a comparison, we confirm the presence of

the various radicals (Zea•+, #Zea•(4), #Zea•(5), #Zea•(9), #Zea•(13), Vio•+, #Vio•(9), #Vio•(13)) and the absence of the #Vio•(4) and #Vio•(5) radicals just as deduced from the ENDOR measurements. HYSCORE spectra (plotted as νR vs νβ) for Zea and Vio radicals on silica-alumina are given in Figures 5 and 6. The R-proton hyperfine coupling tensors were determined from the HYSCORE spectra by analysis of the contour lineshapes of the cross-peaks (Figures 7 and 8) as described earlier.46-48 The cross-peaks plotted on the ν2R(β) and ν2β(R) axes become straight line segments and are plotted in Figures 7A and 8A for Zea and Vio radicals on silica-alumina. The intersection of each segment with the curve (defined by the square of the relation: νR + νβ ) 2 νI) determines two values of the anisotropic hyperfine tensor. These values were assigned to R-proton couplings (MHz) of Zea/Vio radicals obtained by DFT calculations and are indicated by square brackets in Table 3, 4, and 5. Figures 7B and 8B show plots of the coordinates of the HYSCORE ridges of Zea and Vio radicals plotted as the frequencies νR and νβ. Discussion Tables 1 and 2 summarize the isotropic β-methyl proton couplings from the DFT calculations on the carotenoid radical cations and the neutral carotenoid radicals. No methyl hyperfine coupling constants larger than 8-10 MHz were predicted for Car•+ from our DFT calculations, consistent with previous


Focsan et al.

TABLE 4: Anisotropic r-Proton Tensor Components AXX, AYY, and AZZ (MHz) of Zea Neutral Radicals from DFT Calculationsa #Zea•(4) position C2 C2′ C3 C3′ C4 C4′

#Zea•(5)

AXX

AYY

AZZ

Aiso

AXX

AYY

AZZ

Aiso

0.23 -1.16 0.06 -0.15 10.39 -0.93 -0.23 -0.09 -12.55

0.49 -1.04 0.11 -0.10 10.59 -0.62 -0.17 -0.08 -7.94 [-7.04] 3.81 3.41

1.86 0.74 0.40 0.31 12.35 0.92 0.25 0.11 -2.79 [-2.6] 4.6 4.01

0.86 -0.49 0.19 0.02 11.11 -0.21 -0.05 -0.02 -7.76

10.92

7.22

-0.4 -0.43 0.07 -0.19 -0.01 -0.1 -0.29 -0.12 -1.75 -0.74 4.89 4.26 -2.6 -3.16 2.59 [2.6] -14.25 [-15.94] -12.86

-0.37 -0.43 0.14 -0.12 0.09 -0.07 -0.21 -0.11 -1.59 -0.58 4.93 4.4 -1.83 -1.78 2.91

0.29 0.47 0.51 0.4 1.24 0.26 0.32 0.14 -0.89 0.09 5.95 5.18 -0.92 -0.16 5.6 [7.04] -4.74

-0.16 -0.13 0.24 0.03 0.44 0.03 -0.06 -0.03 -1.41 -0.41 5.26 4.61 -1.78 -1.7 3.7 -9.62

-4.07

-8.91

3.78 3.30

4.06 3.57

C5 C7

5.29

C7′

-11.1

C8

-21.66

C8′

2.03

C9 C10

-21.98

C10′ C11 C11′ C12

3.30 [2.6] 6.18 -17.1 [-17.92] -23.4

C12′ C13 C14

4.42 -22.01

C14′ C15 C15′

5.37 6.02 -22.49

5.45 -7.69 [-7.04] -15.51 [-15.94] 2.12

-3.71 [-2.6] -6.99 [-8.26] 4.92

-16.26 [-15.94] 3.89

-17.0 [-15.94] 5.73

-7.6 [-8.26] 7.91 [7.04] 14.43 [15.94] -5.86 [-5.2] -8.08 [-8.26] 10.79

-16.17 [-15.94] 7.19 8.28 -15.83 [-15.94]

-7.84 [-8.26] 13.09 14.83 -7.61 [-8.26]

7.78 [8.26] -11.91

-7.5 -14.72 3.02

2.5 [2.6]

-15.28

-16.82

-9.87 [-8.26] -9.81 2.65 -12.4

C2 C2′ C3 C3′ C4 C4′ C5 C7 C7′ C8 C8′ C9

C10

AXX

AYY

9.65

6.09

9.46

4.4

5.14

10.32

6.62

-11.65 -16.16 6.98 -15.34 8.55 9.71 -15.31

-21.34 [-19.48] -19.4 [-19.48] 5.13 -23.0 [-15.94] 5.77 5.94 -25.28

-14.81

-7.2 [-6.96] -6.63 [-6.96] 12.56

-14.43 6.44 -16.78

-7.72 [-8.26] 13.99 14.0 -8.32 [-6.96]

7.33 7.35 -17.85 [-19.48]

-14.45 -13.49 8.04 -15.83 9.03 9.10 -17.15

#Zea•(13) AZZ

Aiso

-0.03 0.12 0.51 0.40 0.09 0.057 0.31 0.14 -0.08 -0.78 6.03 5.18

-0.13 -0.02 0.24 0.03 0.01 0.001 -0.07 -0.03 -0.15 -0.9 5.34 4.62

0.35 -14.21 [-15.94] -3.67 2.7 [2.6] -18.62 [-17.92] -17.02 [-17.92] -25.02

1.21 -9.87 [-8.26] -3.19 2.76

3.94 -4.81

1.83 -9.63

-1.57 6.63 [7.04] -4.03 [-5.2] -4.42 [-5.2] -7.45 [-8.26]

-2.81 3.94

-17.19 [-15.94]

-11.57

4.62

-0.17 -0.08 0.14 -0.12 -0.02 -0.025 -0.22 -0.1 -0.18 -0.93 5.02 4.41

-11.5

-5.49

4.0

-0.19 -0.1 0.07 -0.19 -0.04 -0.029 -0.3 -0.12 -0.2 -0.99 4.98 4.27

-11.43

3.78

5.03

#Zea•(9) position

6.19 [7.04]

-11.36

AXX -0.12 -0.05 0.08 -0.24 0.01 -0.011 -0.37 -0.16 -0.16 -0.63 6.6 6.4

AYY

AZZ

Aiso

-0.11 -0.02 0.17 -0.15 0.01 -0.006 -0.28 -0.13 -0.11 -0.57 6.65 6.57

-0.08 0.04 0.63 0.51 0.01 0.02 0.41 0.17 -0.07 -0.52 7.96 7.58

-0.1 -0.01 0.29 0.04 0.01 0.001 -0.08 -0.04 -0.11 -0.57 7.07 6.85

0.67 -18.02 [-17.92] -0.36 3.26 [2.6]

0.74 -12.51

-0.69

-0.62

0.25 3.4

1.12 -6.07 [-5.2] 0.23 7.92 [7.04]

0.84 -12.2

-0.35

-0.55

-0.28 4.86

-10.98 -16.55



TABLE 4: Continued #Zea•(9) position

AXX

AYY

#Zea•(13) AZZ

Aiso

AXX

C10′ C11

4.16 4.88

4.92 5.42

7.5 12.45

6.32 7.58

C11′

-21.09

-14.68

-7.25

-14.34

4.92 0.22 [-2.0] -25.52

C12

-23.22

-3.93

5.39

-7.68 [-8.26] 13.03

-16.03

C12′

-17.19 [-15.94] 6.93

8.45

6.01 -22.44

C13

-24.33 C14

-23.66

C14′

6.15

C15

6.39

C15′

-25.48

-17.56 [-15.94] 8.02

-8.3 [-8.26] 14.76

-16.51

-28.44

9.64

6.08

8.4 [8.26] -18.05

15.45 [15.94] -8.58

10.08

5.08

-17.37

-26.96

AYY 5.66 1.35

AZZ

-17.8 [-15.94] -3.45

11.77 4.82 [6.8] -8.64 [-8.26] 1.65

7.43 [8.26] -14.92 [-15.94] -14.95 [-15.94] -19.48 [-19.48] 7.39 [8.26] 5.93

14.67 [15.94] -5.62 [-8.26] -5.3 [-8.26] -8.25 [-6.96] 14.93 [15.94] 14.15

-19.39 [-19.48]

-8.64 [-6.96]

Aiso 7.45 2.13 -17.32 -3.01 9.37 -14.33 -14.86 -18.72 9.47 8.39 -18.33

a HYSCORE couplings (in square brackets) were assigned to these tensors. The bold values are the isotropic coupling constants Aiso given by averaging the three anisotropic coupling tensors AXX, AYY, and AZZ.

Figure 3. Davies ENDOR spectrum of Zea radicals on silicaalumina: (a) experimental. Parameters: T ) 50 K, B ) 3460 G, ν ) 9.691211 GHz, τ ) 200 ns, MW π pulse ) 160 ns, RF π pulse ) 10 µs, SRT ) 1021.02 µs. (b) Simulated spectrum including both isotropic and anisotropic couplings for all five species, (c) simulated spectrum using only the isotropic coupling constants for all five species, (d) simulated spectrum using both isotropic and anisotropic couplings of the radical cation only. The capital letters are discussed in the paper.

calculations.29,31 In contrast, methyl proton hyperfine coupling constants of up to 16 MHz were calculated for the neutral carotenoid radicals #Car•(4), #Car•(5), #Car•(9), and #Car•(13) produced from the corresponding radical cations by proton loss at C4 methylene position or one of the C5, C9, or C13-methyl groups, respectively (see Tables 1 and 2). The DFT calculations showed that the most energetically favorable neutral radical produced by deprotonation of Zea•+ is the one resulting from loss of a proton from position C4. In the case of Vio•+, loss of a C9-methyl proton produces the most energetically favorable neutral radical. The calculations show that loss of a proton from a methyl group at position C5 of Vio•+, which contains an epoxy group at the C5-C6 position, requires higher energy (∼20 kcal/ mol) and results in a nonconjugated neutral allyl radical. Such a radical would exhibit large couplings49,50 which are easy to detect in the EPR spectrum, but no such peaks are observed.

Tables 3, 4, and 5 show the calculated and experimental (HYSCORE values in square brackets) hyperfine coupling tensors of the R-protons. On the basis of our calculations, the hyperfine couplings determined from the HYSCORE analysis of the Zea sample (Table 3 and 4) can be assigned to the Zea•+ R-protons at positions C8 and C8′; to #Zea•(4) at the C4, C7′, C8, C10, C10′, C11, C11′, C12, C14, C15, and C15′; to #Zea•(5) at the C7, C7′, C8′, C10, C11′, C12, C14, and C15′; to #Zea•(9) at the C7′, C8′, C9, C10, C12, C14, and C15; and to #Zea•(13) at the C7′, C8′, C11, C11′, C12′, C13, C14, C14′, and C15′. The hyperfine coupling constants of Vio sample (Tables 3 and 5) can be assigned to the Vio•+ R-protons at positions C7, C7′, C8, C8′, C11, C11′ C15, and C15′; to the #Vio•(9) at positions C7′, C8′, C9, C10′, C11, C11′, C12, C12′, C14, C14′ C15, and C15′; or to the #Vio•(13) at positions C7′, C11, C11′, C12, C12′, C13, C14, C14′, and C15. The DFT calculations predicted large isotropic R-proton hyperfine coupling constants of 14 to 19 MHz in neutral carotenoid radicals. The DFT calculations enabled the spectra to be analyzed, and the spectra showed that a neutral radical is generated by light illumination leading to loss of a proton from the carotenoid radical cation. As a consequence of deprotonation, a change in the unpaired electron spin distribution produces larger methyl proton hyperfine constants for the neutral radical than for the radical cation. As demonstrated by Mairanovsky et al.51 and shown by using simultaneous EPR electrochemistry measurements,52 neutral carotenoid radicals can be produced via deprotonation of carotenoid radical cations and deprotonation and one-electron reduction of dications. Electrochemical experiments with perdeuterated β-carotene,55 which can be oxidized to the radical cation and the closed shell dication, have confirmed the presence of the deprotonation reaction step.53 These experiments showed that the species deprotonated at a methyl group can be reduced in a reverse cyclic voltamogram (CV) resulting in generation of neutral carotenoid radicals. A low potential cathodic peak observed in a reversible CV for a variety of carotenoids has been shown by computer simulation of the CV as due to the neutral radical #Car•.54 Isomerization. Isotropic R-proton hyperfine coupling constants greater than 10 MHz determined from the HYSCORE


Focsan et al.

TABLE 5: Anisotropic r-Proton Tensorcomponents AXX, AYY, and AZZ (MHz) of Vio Neutral Radicals from DFT Calculationsa #Vio•(4) position C2 C2′ C3 C3′ C4 C4′

#Vio•(5)

AXX

AYY

AZZ

Aiso

AXX

AYY

AZZ

Aiso

-3.54 -4.22 0.005 -0.0081 69.67 -8.39 -0.0075 -0.0052 -88.98

-3.22 -3.34 0.01 -0.0081 73.63 -5.10 -0.0055 -0.0042 -55.56

1.23 3.53 0.02 0.0159 84.09 4.25 0.0115 0.0088 -19.66

-1.84 -1.34 0.01 -0.0001 75.80 -3.08 -0.0005 -0.0002 -54.73

-0.003 0.03

-0.002 0.03

-0.63 -0.27 -3.21 0.03

-0.26 -0.20 -1.97 0.05

3.21 -0.08 -0.77 0.13

0.77 -0.18 -1.99 0.07

-0.95 -1.11 -0.002 -0.00061 -0.85 0.08 -0.0064 0.001 -6.92 -2.67 -0.004 0.01 -89.16 -91.81 -1.79 -0.1 -2.42 0.01

-0.84 -1.05 -0.001 -0.0061 -0.49 0.04 -0.0054 0.001 -6.42 -2.06 -0.004 0.01 -56.08 -56.89 -0.98 -0.08 -0.53 0.02

0.89 1.96 0.015 0.0119 2.57 -0.14 0.0106 -0.001 2.63 3.77 0.014 0.02 -19.95 -21.04 3.28 -0.04 2.22 0.06

-0.3 -0.07 0.004 -0.0001 0.41 -0.01 -0.0004 0 -3.57 -0.32 0.002 0.01 -55.06 -56.58 0.17 -0.07 -0.74 0.03

-1.59 0.08 0.34 -0.50 -1.20 0.11

-1.24 0.09 0.45 -0.37 -1.08 0.15

-0.83 0.24 1.25 -0.18 -0.55 0.35

-1.22 0.13 0.68 -0.35 -0.95 0.20

-0.97 0.02 -0.04 -0.17 -0.55 0.03

-0.74 0.02 0.03 -0.15 -0.36 0.05

0.42 0.11 0.76 -0.08 -0.1 0.16

-0.43 0.05 0.25 -0.13 -0.34 0.08

-0.92 0.16 0.22 -0.82

-0.78 0.22 0.3 -0.63

-0.38 0.49 0.67 -0.3

-0.69 0.29 0.4 -0.58

-0.36 0.04 0.05 -0.29

-0.35 0.06 0.08 -0.24

-0.14 0.2 0.32 -0.14

-0.25 0.11 0.15 -0.22

0.022 0.05

0.006 0.04

C5 C7 C7′ C8 C8′ C9 C10 C10′ C11 C11′ C12 C12′ C13 C14 C14′ C15 C15′

#Vio•(9) position C2 C2′ C3 C3′ C4 C4′ C5 C7 C7′

AXX -0.2 -0.08 0.33 -0.19 -0.05 -0.032 -0.15 -0.08 -0.055 -0.32 0.03 1.36

C8 C8′

0.25 -11.9 [-12.22] -3.76 2.01

C9

-22.13 -20.18

C10 C10′

-26.55

C11

3.83 [3.16] 5.1

C11′

20.26

C12

-23.95

C12′

5.09

C13

AYY

#Vio•(13) AZZ

Aiso

-0.18 -0.07 0.38 -0.17 -0.03 -0.027 -0.13 -0.07 -0.041 -0.30 0.05 1.45

-0.04 0.12 0.74 0.33 0.1 0.061 0.25 0.12 0.09 -0.15 0.61 1.84

-0.14 -0.01 0.48 -0.01 0.01 0.001 -0.01 -0.01 -0.002 -0.26 0.23 1.55

1.35 -8.18

4.38 -3.47 [-2.08] -1.66 5.08 [5.06] -5.04 [-5.54] -5.15 [-5.54] -7.59 [-7.44] 9.0

1.99 -7.85

-3.43 2.45 [3.16] -13.6 [-14.1] -13.37 [-14.1] -18.0 [-17.18] 4.34 [5.06] 5.9 [5.54] -14.13 [-14.1] -17.76 [-17.18] 6.42 [5.54]

13.15 [14.1] -6.9 [-5.54] -8.0 [-7.44] 12.28 [14.1]

AXX -0.12 -0.03 0.42 -0.24 -0.004 0.004 -0.2 -0.1 -0.04 -0.16 0.03 1.83

AYY -0.11 -0.01 0.49 -0.22 -0.001 0.002 -0.18 -0.09 -0.02 -0.13 0.07 1.96

AZZ

Aiso

-0.08 0.04 0.95 0.43 0.012 -0.003 0.32 0.16 0.03 -0.08 0.8 2.46

-0.1 -0.003 0.62 -0.01 0.003 0.001 -0.02 -0.01 -0.01 -0.12 0.3 2.08

0.29 15.81 [-14.1] -0.4 2.75

0.88 -10.88 -0.29 3.28

1.56 -4.66 [-5.54] -0.24 6.78

-17.38

-0.85

-0.73

-0.46

-0.68

5.72

4.92

5.53

11.48

7.31

1.69

5.48 [6.56] -8.65 [-7.44] -2.04

2.51

-2.95 3.18

0.91 -10.45 -0.31 4.27

-13.59 -12.9

8.17 -13.76 -16.57 7.93

0.36 [-0.32] -25.45 -4.38 [-5.06] 6.15 [5.54] -25.38 -27.58

-17.83 [-17.18] -3.9 [-3.16] 7.54 -16.79 [-17.18] -17.09 [-17.18]

14.75 [14.1] -6.41 [-7.44] -6.33 [-7.44]

-17.31 -3.44 9.48 -16.19 -17.0



TABLE 5: Continued #Vio•(9)

#Vio•(13)

position

AXX

AYY

AZZ

Aiso

AXX

AYY

AZZ

Aiso

C14

-23.99

-30.05

-20.46

-8.55

-19.69

5.99 [5.54] 6.39 [5.54] -25.4

-8.45 [-7.44] 14.47 [14.1] 15.57 [14.1] -8.57 [-7.44]

-16.75

C14′

-17.81 [-17.18] 7.8

7.87

15.62 [14.1] 15.33 [14.1] -9.05

9.97

C15 C15′

8.45 -18.02 [-17.18]

9.42 10.14 -17.33

6.43 [5.54] 5.50 [5.54] -27.83

6.62 -20.1

9.15 -18.99

a HYSCORE couplings (in square brackets) were assigned to these tensors. The bold values are the isotropic coupling constants Aiso given by averaging the three anisotropic coupling tensors AXX, AYY, and AZZ.

Figure 4. Davies ENDOR spectrum of Vio radicals on silicaalumina: (a) experimental. Parameters: T ) 40 K, B ) 3460 G, ν ) 9.686928 GHz, τ ) 200 ns, MW π pulse ) 80 ns, RF π pulse ) 20 µs, repetition time ) 1021.02 µs. (b) Simulated spectrum including both isotropic and anisotropic couplings for all three species, (c) simulated spectrum using only the isotropic coupling constants for all three species, (d) simulated spectrum using both isotropic and anisotropic couplings of the radical cation only. The capital letters are discussed in the paper.

analysis have been previously reported for the radical cations of canthaxanthin on a silica-alumina support.27 Such large coupling constants could be due to the presence of different isomers in the radical cation other than the trans isomer considered above. The role of isomerization was supported by other experiments55-60 which showed that carotenoids isomerize upon electron transfer when the radical cations or dications are formed. For example, trans-to-cis isomerization can occur by electrochemical oxidation in CH2Cl2,56 bulk electrolysis, or chemical oxidation with FeCl3 in CH2Cl2,57,60 in the presence of traces of acids in CH2Cl2,55,58 or in the presence of semiconductors like CdS or ZnO particles that catalyze the photoisomerization.59 Isomerization can occur because, during the formation of the radical cation, the alternating single and double bond lengths shorten and lengthen respectively to extended radicals, which reduces the barrier to isomerization about the original double bonds. For example, the rotation barrier in C2H4 is 65.6 kcal/ mol61 and that in the allyl radical has been measured to be 15.7 ( 1.0 kcal/mol62 and calculated to be 17.4 kcal/mol with the B3LYP functional at the DFT level.63 Upon formation of a dication58 by a two-electron transfer, the change in geometry is even greater which should reduce the barrier to isomerization even further. Since isomerization can occur, we calculated the isotropic methyl coupling constants (shown to contribute most to the ENDOR spectra in Figures 3 and 4) of some cis and di-cis isomers of Zea and Vio radical cations. As shown in Table 6, upon isomerization, the isotropic proton coupling constants

change for the particular methyl group; some couplings decrease and other increase, but none exceed 10 MHz, except for 9-cis Zea•+ which exhibits a coupling of 10.84 MHz. These additional isomers are higher in energy than the corresponding all-trans isomer (see Table 6) so isomerization is unlikely except possibly for 9-cis Zea•+ (1.4 kcal/mol higher), 13-cis Zea•+ (2 kcal/mol higher), and 15-cis Zea•+ (3.4 kcal/mol higher). However, even if isomerization does occur, it cannot account for the presence of larger isotropic methyl coupling constants of 12 to 16 MHz which are only predicted to occur for the carotenoid neutral radicals #Car•. Photoprotection. There are a number of different photoprotection mechanisms at work in photosynthetic systems.64-71 One class, nonphotochemical quenching (NPQ), involves the dissipation of energy absorbed in the light harvesting apparatus into thermal energy. Some of the NPQ mechanisms involve carotenoids of the xanthophyll cycle, specifically, Zea and Vio. One major NPQ route takes place in the light harvesting complex II (LHC-II), and another takes place in the highly homologous protein PsbS.69-71 Recent crystal structures of LHCII72,73 have led to proposed mechanisms for how NPQ functions in LHC-II. Both proposed mechanisms involve the replacement of Vio by Zea in LHC-II under high light conditions. The presence of Zea leads to dissipation of excess excitations in LHC-II. Although both proposed mechanisms are fairly detailed, neither takes into account the formation of the charge-transfer complex of reduced chlorophyll, radical anion Chl•-, and oxidized zeaxanthin, radical cation Zea•+, observed by the Fleming group.33 In addition, neither model addresses the subsequent deprotonation of Car•+ to produce #Car• described above. The loss of protons from the C4(4′) methylene position and C5(5′)-, C9(9′)-, or C13(13′)-methyl groups of Car•+ has potential importance in NPQ in the light harvesting complex LHC-II (and in the homologous protein PsbS) through the xanthophyll cycle. The reactions in Scheme 3 need to be considered in any NPQ mechanism involving Zea (together with analogous reactions for Vio). In the first reaction, excitation of Zea or a nearby Chl, either by direct absorption of light or by transfer of excitation energy from other pigments in LHC-II, produces, with some finite probability, the charge-transfer state Zea•+‚‚‚Chl•- observed by the Fleming group.33 Our experiments on silicaalumina suggest that further direct excitation or arrival of a second excitation in LHC-II would cause Zea•+ to deprotonate to form #Zea•, while our previous electrochemical measurements74,75 on other carotenoids suggest that deprotonation may occur spontaneously in the presence of water or other proton acceptors. In the context of NPQ, formation of the neutral radical would have two significant consequences. First, collapse of the charge


Figure 5. Contour plot of the HYSCORE spectrum of Zea radicals on silica-alumina: (A) Parameters: T ) 40 K, B ) 3450 G, ν ) 9.672245 GHz, τ ) 140 ns, repetition time ) 799.68 µs. The spectrum was obtained after Fourier transformation of 2D time-domain patterns containing 160 × 160 points for the dataset. Data manipulation before 2D Fourier transformation: baseline correction followed by SinBell windowing, zero-field filling to 1024 points. (B) T ) 40 K, B ) 3422 G, ν ) 9.672145 GHz, τ ) 172 ns, repetition time ) 799.68 µs. The spectrum was obtained after Fourier transformation of 2D time-domain patterns containing 160 × 160 points for the dataset. Data manipulation before 2D Fourier transformation: baseline correction followed by LorGauss windowing, zero-field filling to 1024 points.

transfer state to regenerate Zea and Chl is blocked because the product would be the conjugate base of Zea, a highly unstable product. Once formed, #Zea• should have a fairly long lifetime in LHC-II. Second, free radicals are very efficient quenchers of both singlet and triplet excited states, so that #Zea• would be a very efficient quencher of excitation in LHC-II. The neutral radical of Zea would effectively shut down the input of energy to PSII. It has been established that photoprotection by NPQ occurs when LHC-II contains Zea but not Vio. Although Zea has a

Focsan et al.

Figure 6. Contour plot of the HYSCORE spectrum of Vio radicals on silica-alumina: (A) Parameters: T ) 40 K, B ) 3422 G, ν ) 9.590056 GHz, τ ) 172 ns, repetition time ) 999.6 µs. (B) T ) 40 K, B ) 3422G, ν ) 9.590080 GHz, τ ) 140 ns, repetition time ) 999.6 µs. The two spectra for Vio radicals were obtained after Fourier transformation of 2D time-domain patterns containing 160 × 160 points for the dataset. Data manipulation before 2D Fourier transformation: baseline correction followed by SinBell windowing, zero-field filling to 1024 points.

somewhat lower oxidation potential (571mV vs SCE) than Vio (681 mV vs SCE)76,76 both can form the radical cation on silicaalumina matrix and both can form the neutral radical upon light irradiation, leading to the important question as to why only Zea is photoprotective. There is certainly sufficient energy with both Zea and Vio to form the charge-transfer complex observed by the Fleming group.33 However, close examination of the crystal structure of LHC-II suggests that the deprotonation of Vio•+ may not be possible in the protein. The binding site for Zea/Vio in LHC-II is highly nonpolar along most of its length with hydrocarbons and nonpolar amino acid side groups surrounding the long, conjugated chain. The molecule is positioned by hydrogen bonds to the terminal -OH groups. There are no proton acceptors to receive the proton from the C9(9′)- or C13(13′)-methyl groups, so that deprotonation at those


Figure 7. (A) Plots of the coordinates of the HYSCORE ridges of Zea radicals plotted as the squares of the frequencies ν2R and ν2β with least-squares fits to straight lines in those coordinates. The intersection of these straight lines (light blue, orange, red, dark blue, green, brown) with the black curve defined by (νR + νβ)2 ) (2νI)2, where νI is the proton frequency, give two principal values of hyperfine coupling tensor A, as (A/2 ) νR(β) - νI and (A/2 ) νI - νβ(R). The colors correspond as follows: light blue to (6.96 and (19.48 MHz tensors, orange to (5.2 and (17.92, red to (8.26 and (15.94, dark blue to (2.6 and (7.04, green to -1.58 and (6.34, and brown to -2.0 and (6.8. These tensors were assigned in Tables 3 and 4 to different R-proton coupling tensors on the basis of DFT calculations. (B) Plots of the coordinates of the HYSCORE ridges of Zea radicals plotted as the frequencies νR and νβ. The straight lines become the colored corresponding curves, and the black curve becomes a line defined by νR + νβ ) 2νI. Because the HYSCORE spectra for Zea radicals were taken at two different magnetic fields (B ) 3450 G and B ) 3422 G) we used the field correction described in reference 49.

positions would be energetically highly unfavorable. The headgroups are in a more polar environment near the surface of the membrane. The ring is in contact with the aqueous environment outside the membrane and with the polar headgroup of a tightly bound phospholipid. This environment would readily accept the proton from the C4(4′) methylene position or from the C5(5′)-methyl group. Although photoexcitation is necessary to deprotonate the radical cation on silica-alumina, our


Figure 8. (A) Plots of the coordinates of the HYSCORE ridges of Vio radicals obtained as described in caption of Figure 7. The colors correspond as follows: dark blue to (7.44 and (17.18 MHz tensors, green to (5.54 and (14.1, orange to -2.08 and (12.22, red to (0.32 and (6.56, and brown to (3.16 and (5.06. These tensors were assigned in Tables 3 and 5 to different R-proton coupling tensors on the basis of DFT calculations. (B) Plots of the coordinates of the HYSCORE ridges of Vio radicals plotted as the frequencies νR and νβ. The straight lines become the colored corresponding curves, and the black curve becomes a line defined by νR + νβ ) 2νI.

electrochemical measurements76,77 show that the radical cation of a wide range of carotenoids have pK’s ranging between 4 and 7 and Zea•+ could deprotonate spontaneously. Whereas loss of the C4(4′)-methylene proton or the C5(5′)methyl proton is favored for Zea, the epoxide group blocks that loss in Vio. Even our UV irradiation of 350 nm is insufficient to cause loss of a proton from the C4(4′) position or C5(5′)methyl group of Vio•+. The usual absorption of the radical cation is around 900 nm,33,77 and 350 nm light provides almost 50 kcal/mol of excess energy which is larger than the energy differences of 10 kcal/mol for the neutral radicals of Zea and of 20 kcal/mol for Vio. Clearly, not all of the excess energy is available for radical isomer formation as #Vio•(4) and #Vio•(5) are not formed. In photosynthesis, the photons or excitation are substantially lower in energy, and consequently, the loss of


Focsan et al.

TABLE 6: Calculated Isotropic β-Methyl Proton Hyperfine Couplings Aiso (MHz) for Some cis and di-cis Isomers of Zeaxanthin and Violaxanthin Radical Cations and Their Relative Energies (kcal/mol) to All-Trans Radical Cations a position C1-CH3 C1′ -CH3 C5-CH3 C5′-CH3 C9-CH3 C9′-CH3 C13-CH3 C13′-CH3 a

9-cis Zea·•+

13-cis Zea·•+

15-cis Zea·•+

11,11'-dicis Zea·•+

11,15'-dicis Zea·•+

11,11'-dicis Vio•+

11,15'-dicis Vio•+

∆E ) 1.4 0.09 -0.06 -0.05 -0.03 4.46 7.13 10.84 8.23 5.65 4.6

∆E )2.0 0.07 -0.07 -0.05 -0.02 4.99 7.25 7.44 8.37 7.29 4.69

∆E ) 3.4 0.07 -0.08 -0.04 -0.03 5.65 6.71 8.15 7.99 4.7 4.4

∆E ) 10.6 0.08 -0.08 -0.04 -0.02 5.58 6.59 8.16 7.99 6.72 6.4

∆E ) 8.8 0.06 -0.08 -0.04 -0.02 5.48 6.75 7.9 7.93 6.28 4.28

∆E ) 10.2 0.11 0.06 0.03 0.22 0.25 0.22 9.78 9.77 7.82 7.75

∆E ) 8.5 0.11 0.06 0.04 0.23 0.25 0.23 9.51 9.72 7.29 5.5

Etrans ) -1707.353479 au for Zea•+; Etrans ) -1857.749206 au for Vio•+.

SCHEME 3: Modified Schematic Representation of the Vio/Zea Equilibrium

a proton from the C4(4′)-methylene position or from the C5(5′)-methyl group of Vio•+ will be even more unlikely. Only when Zea is present in LHC-II would a neutral radical be formed by deprotonation at the C4(4′)-methylene position or C5(5′)-methyl position; the resulting neutral radical would act as a highly effective trap and quench all excitations passing through LHC-II. When Vio is present in LHC-II, formation of the neutral radical is not possible so that collapse of the charge transfer state would be rapid, restoring the LHC-II back to its initial unquenched state and allowing excitations to flow freely to PSII. Thus, because the charge transfer state is observed in intact thylakoids membranes in the quenched state, the subsequent deprotonation of the radical cation must be considered in any discussion of NPQ. Deprotonation offers a simple mechanism based on the structure of LHC-II for quenching and photoprotection of PSII and a simple explanation for the differential effects of Zea and Vio in LHC-II.

deprotonation, formation of zeaxanthin neutral radical via loss of the C4(4′)-methylene proton or the C5(5′)-methyl proton is energetically most favorable. In contrast, proton abstraction from C4(4′) position or C5(5′)-methyl group in violaxanthin requires very high energy, and loss of the C9(9′)-methyl proton requires the least amount of energy. These results provide new insights into the role of proton transfer and loss in the photophysical processes that underlie photoprotection in photosynthetic systems. Acknowledgment. This work was supported in part by the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Sciences, U.S. Department of Energy, Grant DE-FG02-86ER-13465, and the catalysis center program. The Hungarian part of the study was supported by the grant from OTKA K 60121 (Hungarian National Research Foundation). This work was carried out in part using the resources provided by the Alabama Supercomputer Center. D.A.D. is indebted to the Robert Ramsay Endowment of The University of Alabama. The work was performed in part at the EMSL (Pacific Northwest National Laboratory), a national scientific user facility sponsored by the U.S. Department of Energy. Nikolay Polyakov is thanked for discussions. Supporting Information Available: Echo-induced fieldswept EPR spectrum of zeaxanthin radicals generated by irradiation at 77 K on silica-alumina, Fourier transform of the 2-pulse ESEEM spectrum of zeaxanthin radicals on silicaalumina, Mims ENDOR spectra of zeaxanthin and violaxanthin radicals on silica-alumina, optimized x, y, z coordinates at B3LYP/6-31G (d, p) level for Zea•+, #Zea•(4), #Zea•(5), #Zea•(9), #Zea•(13), Vio•+, #Vio•(4), #Vio•(5), #Vio•(9), and #Vio•(13). This material is available free of charge via the Internet at http://pubs.acs.org.

Conclusions Pulsed EPR spectroscopy was used to characterize radical species produced upon photo-oxidation of natural zeaxanthin and violaxanthin on silica-alumina. The isotropic methyl proton hyperfine coupling constants obtained from the Davies pulse sequence spectra and anisotropic R-proton hyperfine coupling constants determined from a 2D-HYSCORE experiment were assigned on the basis of DFT calculations with the B3LYP exchange-correlation functional. The calculations performed in this study and our previous work31 demonstrated that such proton methyl couplings are larger for carotenoid neutral radicals. The neutral radicals (#Car•) can be produced as a result of proton loss from the methyl groups of positions C5(5′), C9(9′), or C13(13′) or from the C4(4′)-methylene position of the radical cation Car•+. According to the calculated energies of the Car•+

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Pulsed EPR and DFT Characterization of Radicals Produced by Photo

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